Programmable oligonucleotide synthesis

ABSTRACT

The invention relates to methods and devices for preparing synthetic nucleic acids.

This application is divisional of U.S. Ser. No. 12/438,425, file Feb.23, 2009 now U.S. Pat. No. 8,173,368, which is a 35 U.S.C. 371 NationalPhase Entry Application from PCT/EP2007/007417, filed Aug. 23, 2007,which claims the benefit of German Patent Application No. 10 2006 0394798.8 filed on Aug. 23, 2006, the disclosure of which is incorporatedherein in its entirety by reference.

DESCRIPTION

The invention relates to methods and devices for preparing syntheticnucleic acids.

INTRODUCTION

There is a high demand for synthetic nucleic acids in molecular biologyand biomedical research and development. Synthetic nucleic acids (DNA,RNA or their analogues) are mainly prepared using column-basedsynthesizers.

Particularly important and widespread applications for synthetic nucleicacid polymers are primers for the polymerase chain reaction (PCR)(Critical Reviews in Biochemistry and Molecular Biology 26 (3/4),301-334, 1991) and the sequencing method according to Sanger (Proc. Nat.Acad. Sci. 74, 5463-5467, 1977).

Synthetic DNA also has a role in the preparation of synthetic genes.Methods of gene synthesis are described for example in U.S. 6,586,211B1, in PCT/EP2004/013131, in WO 00/13017 A2, in S. Rayner et al., PCRMethods and Applications 8 (7), 741-747, 1998, in WO 90/00626 A1, in EP385 410 A2, in WO 94/12632 A1, in WO 95/17413 A1, in EP 316 018 A2, inEP 022 242 A2, in L. E. Sindelar and J. M. Jaklevic, Nucl. Acids Res.(6), 982-987, 1995, in D. A. Lashkari, Proc. Nat. Acad. Sci. USA 92(17), 7912-7915, 1995, and in WO 99/14318 A1, which are incorporated asreference.

Another two fields of application with increasing demand are theproduction of microarrays or biochips from oligonucleotide probes (1.Nature Genetics, Vol. 21, Supplement (complete), January 1999, 2. NatureBiotechnology, Vol. 16, 981-983, October 1998, 3. Trends inBiotechnology, Vol. 16, 301-306, July 1998) and the preparation ofinterfering RNA (iRNA or RNAi) for the modulation of gene expression intarget cells (PCT/EP01/13968).

The aforesaid fields of application of molecular biology providevaluable contributions in the development of active compounds, theproduction of active compounds, combinatorial biosynthesis (antibodies,effectors such as growth factors, neurotransmitters etc.), inbiotechnology (e.g. enzyme design, pharming, biological productionmethods, bioreactors etc.), in molecular medicine in tissue engineering,in the development and application of new materials (e.g. materials suchas spider silk and mother of pearl), in the development and use ofdiagnostic agents (microarrays, receptors and antibodies, enzyme designetc.) or in environmental engineering (specialized or tailor-mademicroorganisms, production methods, remediation, sensors etc.). Themethod according to the invention can thus be employed in all theseareas.

PRIOR ART

The commonest method for the preparation of synthetic nucleic acids isbased on the fundamental work of Caruthers and is known as thephosphitamide method (M. H. Caruthers, Methods in Enzymology 154,287-313, 1987). The sequence of the resultant molecules can becontrolled by the order of synthesis. Other methods, such as theH-phosphonate method, serve the same purpose of successive synthesis ofa polymer from its subunits, but have not found such widespreadapplication as the method according to Caruthers.

To make it possible to automate the chemical method of polymer synthesisfrom subunits, solid phases are generally employed, on which the growingmolecular chain is anchored. On completion of synthesis it is split off,which requires a suitable linker between the actual polymer and thesolid phase. For automation, as a rule the method employs solid phasesin the form of activated particles, which are packed in a column, e.g.controlled pore glass (CPG). These solid phases as a rule only carry onespecifically removable type of oligo with a programmed sequence. Theindividual synthesis reagents are then added in a controllable manner inan automatic machine, which mainly provides the automated addition ofthe individual reagents to the solid phase. The quantity of moleculessynthesized can be controlled by the amount of support material and thesize of the reaction batches. For the aforementionedmolecular-biological methods, these amounts are either sufficient oreven too high (e.g. in the case of PCR primers). Some degree of paralleloperation for production of a multiplicity of different sequences isachieved through arranging several columns in an assembly of apparatus.Thus, equipment with 96 parallel columns is known by a person skilled inthe art.

A variant and further development for the production of syntheticnucleic acids is the in situ synthesis of microarrays (array arrangementof nucleic acids in a matrix). This is carried out on a substrate thatis loaded with a multiplicity of different sequences during thesynthesis. The great advantage of the in situ synthesis methods formicroarrays is the preparation of a multiplicity of oligomers ofdifferent and defined sequence at addressable locations on a commonsupport. The synthesis has recourse to a manageable set of feedmaterials (in the case of DNA microarrays, as a rule the 4 bases A, G, Tand C) and from these it builds up any sequences of nucleic acidpolymers.

The individual molecular species can be demarcated on the one hand byseparate fluidic compartments for addition of the synthesis feedmaterials, as is the case e.g. in the so-called in situ spotting methodor piezoelectric techniques, based on inkjet printing technology (A.Blanchard, in Genetic Engineering, Principles and Methods, Vol. 20, Ed.J. Sedlow, 111-124, Plenum Press; A. P. Blanchard, R. J. Kaiser, L. E.Hood, High-Density Oligonucleotide Arrays, Biosens. & Bioelectronics 11,687, 1996).

An alternative method is the spatially-resolved activation of synthesissites, which is possible through selective illumination, throughselective or spatially-resolved generation of activation reagents(deprotection reagents) or through selective addition of activationreagents (deprotection reagents).

Examples of the methods known to date for the in situ synthesis ofmicroarrays are

-   -   photolithographic light-based synthesis (McGall, G. et al.; J.        Amer. Chem. Soc. 119; 5081-5090; 1997),    -   projector-based light-based synthesis (PCT/EP99/06317),    -   fluidic synthesis by means of physical separation of the        reaction spaces (known by a person skilled in the art from the        work of Prof. E. Southern, Oxford, UK, and of the company Oxford        Gene Technologies, Oxford, UK),    -   indirect projector-based light-controlled synthesis by        light-activated photo-acids and suitable reaction chambers or        physically separated reaction spaces in a reaction support,    -   electronically induced synthesis by spatially-resolved        deprotection on individual electrodes on the support using        proton production induced by the electrodes (known from, among        others, the products of the company Combimatrix) and    -   fluidic synthesis by spatially-resolved deposition of the        activated synthesis monomers (known from A. Blanchard, in        Genetic Engineering, Principles and Methods, Vol. 20, Ed. J.        Sedlow, 111-124, Plenum Press; A. P. Blanchard, R. J.        Kaiser, L. E. Hood, High-Density Oligonucleotide Arrays,        Biosens. & Bioelectronics 11, 687, 1996).

Methods of preparation of synthetic nucleic acids, in particular nucleicacid double strands on a common solid support, are also known from WO00/49142 and WO 2005/051970.

OBJECT OF THE INVENTION

An improved method is to be provided for the preparation of syntheticnucleic acids of optional sequence, in particular nucleic acid doublestrands, through the preparation of suitable solid-phase-supportedsynthetic libraries. Moreover, an improved method is to be provided forthe preparation of synthetic nucleic acids of optional sequence, inparticular nucleic acid double strands, through the preparation ofsuitable solid-phase-supported synthetic libraries and the subsequentjoining together of at least two nucleic acid fragments from the librarythrough binding or covalent linkage of these two nucleic acid fragmentsto one another, wherein preparation of the library includes control ofthe quantitative proportions of the constituents of the library to oneanother.

The nucleic acid fragments are joined together preferably by a specifichybridization reaction between overlapping regions of mutuallycomplementary segments of the nucleic acid fragments, thereby obtaininglonger synthetic double-stranded nucleic acids. The individual sequencesegments used for building up longer nucleic acids preferably have alength of 20-100 or 20-300 nucleotide building blocks, preferably of25-50 or 25-100 nucleotide building blocks, for example about 30nucleotide building blocks. The sequence segments are preferablyselected in such a way that they at least partially overlap a sequencesegment of the antisense strand of the complementary nucleic acid thatis to be synthesized, so that the nucleic acid strand to be synthesizedcan be built up by hybridization of individual sequence segments. In analternative embodiment, the sequence segments are preferably selected sothat the sequence segments on both strands of the nucleic acid to besynthesized completely overlap, and accordingly preparation of a more orless complete double strand now only requires covalent linkage of thephosphodiester backbone. The length of the complementary regions oroverlaps between individual fragments is e.g. 10-50 or 10-100 nucleotidebuilding blocks, preferably 12-25 or 20-80 nucleotide building blocks,especially preferably about 15-20 nucleotide building blocks and mostpreferably about 15 or about 20 nucleotide building blocks. If theoverlapping or complementarity region between two nucleic acid fragmentshas a high AT content, e.g. an AT content >50%, preferably an ATcontent >60%, especially preferably an AT content >65%, the bindingconstant is lower in comparison with GC-richer sequences. Accordingly,for thermodynamic reasons, hybridization between these fragments may beof comparatively low efficiency. This can have an influence on theassembly of 2 or more fragments. A possible sequence-dependentconsequence is a reduced yield of nucleic acid double strands with thecorrect target sequence.

One aim of the method according to the invention is to influence thethermodynamic relations during assembly from 2 or more fragments bycontrolling or by controlling and regulating the quantitativeproportions of the fragments in a reaction batch, in order to improvethe yield of correct nucleic acid double strands. In particular, thethermodynamic parameters are modulated in a reaction for binding atleast 2 nucleic acid fragments to one another. Modulation of thethermodynamic parameters means, in particular, that the binding of thetwo nucleic acid fragments to one another, which is subject to the lawof mass action, is improved. It is especially preferable for themodulation of the thermodynamic parameters in the reaction to comprisecontrol of the quantitative proportions of individual nucleic acidfragments, in particular through the use of larger amounts of nucleicacid fragments that have a high proportion of AT. If at least somenucleic acid fragments, for modulation of their thermodynamic parametersin the reaction of at least 2 nucleic acid fragments, are used in anincreased amount relative to other fragments, this can be achieved forexample in that at least some nucleic acid fragments, which have a highAT content, are used in an increased amount relative to other fragments.

By controlling the quantitative proportions of individual nucleic acidfragments, in particular by using larger amounts of nucleic acidfragments that have a high proportion of AT, the yield of correcthybridization products and therefore also the yield of correct nucleicacid double strands can be improved. The quantity of the population ofthe corresponding nucleic acid fragments can thus be improved bypreferably ≧10%, especially preferably ≧50% or even more, e.g. by up toa factor of 100 or 1000 relative to other nucleic acid fragments withouta high proportion of AT.

Apart from the proportion of AT, there are also other parameters thathave an influence on the yield of target sequences. These include thevarying synthesis efficiency of the individual fragments oroligonucleotides during extension in the synthesis process. A personskilled in the art knows, for example, that building block G inphosphoramidite methods couples at lower yield to the polymer strandthat is to be extended than the other nucleotide building blocks.Moreover, a person skilled in the art is aware of dependences of thesynthesis efficiency on the complete sequence of the polymer strand thatare empirically evident, but not in every case already predictable. Thisincludes for example the synthesis of several G building blocks insuccession.

These deviations in the availability or kinetics of individual fragmentsfor the assembly of 2 or more fragments into a target sequence can alsobe influenced by controlling or controlling and regulating thequantitative proportions. The deviation may be well known and it may bepossible to calculate it, or it may only be known empirically andobserved in experiments. Accordingly, the method according to theinvention can be optimized e.g. iteratively with measurement of theresult, for example the yield of target sequence. One embodiment of theinvention is the use of a stored-program device, in order to control thepredicted optimal composition of the quantitative proportions on thebasis of known regularities for new fragment sequences and targetsequences. During reaction this can take place by means of a computer orsimilar control equipment. The influencing factors and settings can berecorded in a database, which in one embodiment is contained in astored-program device and is used directly or indirectly in the controlof the synthesis.

The reaction products of a library synthesis are characterized byconsiderable variety of the sequences, programming of which is freelyselectable during the synthesis operation. A numerical example willillustrate the great variety of such a library. A microarray from theGENIOM® system, for which the nucleic acid molecule populations aresynthesized on individual synthesis locations in a special microfluidicsupport, can for example (status in the year 2006) synthesize up to 60000 freely selectable oligonucleotides with a sequence of up to 60nucleotides. The equipment provides spatially-resolved synthesis of thenucleic acids using a projector-based method (see e.g. WO 00/13018 or WO00/13017).

The aim of the improved method is to provide nucleic acids with high andrationally programmable diversity of the sequences and controllablequantitative proportions of the individual sequence representatives orfragments (constituents of the library) for subsequent processes in anext step.

Examples of subsequent processes, for which the invention can be used,are:

-   -   production of nucleic acid fragments as primers for primer        extension methods, strand displacement amplification, polymerase        chain reaction, site directed mutagenesis or rolling circle        amplification,    -   production of synthetic genes, gene fragments, gene clusters,        gene transfers, gene vectors, chromosomes, genomes, optimized        genomes, minimal gene clusters, minimal genomes, completely        synthetic genomes or of mixtures with directed or randomized        variants of synthetic genes, gene fragments, gene clusters, gene        transfers, gene vectors, chromosomes, genomes, optimized        genomes, minimal gene clusters, minimal genomes, completely        synthetic genomes,    -   modulation of gene expression by means of RNAi or antisense        methods, in which one more copy of the nucleic acid or nucleic        acids produced can be provided by an RNA polymerase,    -   production, extraction, purification, isolation or preparation        of analytes (sample preparation) for the logically subordinate        analysis by microarrays, by sequencing methods, by parallel        sequencing methods, by amplification methods (strand        displacement amplification, polymerase chain reaction or rolling        circle amplification) or analysis in gel electrophoresis,    -   RNA libraries with e.g. 2 or more sequences for translation in        vitro or in vivo,    -   cloning of the nucleic acids produced alone or in combination        with further sequences by means of vectors or plasmids,    -   production of minimal genomes, optimized genomes, reduced        genomes, mixed genomes of various species, wherein the whole        planned stock of the genome or a portion thereof can be encoded        by the synthetic nucleic acids,    -   production of target organisms with permanently or temporarily        integrated, transformed, transfected or otherwise inserted        synthetic genes, gene fragments, gene clusters, gene transfers,        gene vectors, chromosomes, genomes, optimized genomes, minimal        gene clusters, minimal genomes, completely synthetic genomes or        of mixtures with directed or randomized variants of synthetic        genes, gene fragments, gene clusters, gene transfers, gene        vectors, chromosomes, genomes, optimized genomes, minimal gene        clusters, minimal genomes, completely synthetic genomes,    -   production of the target organisms described in the preceding        item for the improvement, change or reduction of a native        substance occurring in the target organism, e.g. an amino acid        chain, a protein, an organic substance, a hydrocarbon, a drug or        a precursor thereof, a nucleic acid, a pheromone, or some other        substance for the provision of an article used by humans or for        production of a substance that is then put to further use,    -   ligation of the nucleic acids in vectors, YACs, BACs,        chromosomes or plasmids,    -   validation or testing of hybridization assays and associated        reagents and kits by means of the nucleic acid polymers        produced, in the areas of microarrays, biochips, dot blots,        Southern or Northern blots, bead arrays, serial analysis of gene        expression (SAGE), PCR, real-time PCR,    -   reference or calibration methods or steps within assays from the        areas of microarrays, dot blots, Southern or Northern blots,        bead arrays, serial analysis of gene expression (SAGE), PCR,        real-time PCR,    -   production of binding agents such as aptamers and ribozymes and        indirect production via the translation in vivo or in vitro of        peptides, proteins, antibodies, antibody fragments, peptides        acting analogously to antibodies, proteins acting analogously to        antibodies,    -   production, via translation in vivo or in vitro of        glycoproteins, proteoglycans or complexes with optionally        peptide fraction, protein fraction, RNA fraction or DNA        fraction, such as ribosomes or proteasomes.

DETAILED DESCRIPTION OF THE INVENTION AND EMBODIMENTS THEREOF

Preferred methods of production of synthetic nucleic acids from a solidsupport are known from WO 00/49142 and WO 2005/051970. Reference is madeexpressly to the contents of these documents, and they are incorporatedherein in their entirety.

Preferably, in the method according to the invention, the syntheticnucleic acids are prepared by synthesizing a multiplicity of differentnucleic acid fragments at various positions of a common solid support.Preferably the synthesis of the nucleic acid fragments comprisesconstruction from nucleotide building blocks by wet-chemical and/orphotochemical methods on the support, subsequent detachment of thenucleic acid fragments and assembling of the fragments to the desirednucleic acid double strand. Furthermore, the synthesis can includeamplification steps, in which the synthesized nucleic acid fragmentsor/and optionally double-stranded intermediates formed from them aresubmitted to amplification, e.g. PCR. For this purpose, nucleotidebuilding blocks and an enzyme that brings about amplification can beadded. Amplifications can take place on the support, i.e. before or/andafter detaching the nucleic acid fragments, or/and after elution fromthe support.

The support can be selected from flat supports, porous supports,reaction supports with electrodes, reaction supports with particles orbeads, microfluidic reaction supports, which optionally have surfacemodifications such as gels, linkers, spacers, polymers, amorphous layersor/and 3D matrices, and combinations of the aforesaid supports.Preferably the support is a microfluidic support.

The nucleic acid fragments are preferably produced by spatially or/andtime-resolved in situ synthesis on the support, for example by spatiallyor/and time-resolved illumination by a programmable light source matrix.The spatially or/and time-resolved synthesis can take place in amicrofluidic support with one or more fluidic reaction spaces and one ormore reaction regions within a fluidic reaction space.

Different amounts of nucleic acid fragment species used for assembly canbe produced by using several regions and/or larger regions for thesynthesis of the particular nucleic acid fragments on the support. Anappropriately modified support is also an object of the invention.

A further—optionally independent—object of the invention comprisescarrying out the assembly of nucleic acid fragments to nucleic aciddouble strands in several steps. In a first step, the nucleic acidfragments synthesized on the support are provided at the 5′- or/and3′-end with one or more generic primer sequences of preferably 10-20 or10-100 bases, especially preferably of 10-30 bases, even more preferablyabout 15 bases, with the primer sequences being selected so thatamplification is possible directly for the individual fragment, for aproportion of all fragments in a mixture, for all fragments in a mixtureor after hybridization of two or more nucleic acid fragments withpartial complementary sequence. After cleaving off the nucleic acidfragments provided with primers from the support and optionally afterelution from the support and optionally a hybridization of fragmentpairs with complementary sequence, a subsequent amplification takesplace, e.g. by PCR, by adding corresponding primers. In theamplification reaction there is formation of nucleic acid fragments thatcontain the generic primer sequence at their ends. After cleaving-offthe primer sequence, e.g. by means of restriction endonucleases, theresultant nucleic acid fragments can be submitted to furtheramplification cycles, in order to produce a nucleic acid double strand.

In one embodiment, the nucleic acid double strand produced by synthesisof fragments and their subsequent assembly is inserted into a vector,e.g. a plasmid, and transferred into a suitable host cell, e.g. abacterial cell.

The preparation of the nucleic acid polymers offers, at several pointsof the method, the possibility of introducing modifications or labelinginto the reaction products by known methods. This includes labelednucleotides, which are modified e.g. with haptens or optical markers,such as fluorophores and luminescence markers, labeled primers ornucleic acid analogues with special properties, such as special meltingpoint or accessibility for enzymes. Embodiments of the invention cantherefore include the following functions and methods and can make theassociated laboratory processes possible:

-   -   labeling of the oligonucleotides, of the fragments, of        covalently or noncovalently bound hybrid strands constructed        therefrom and of the target sequences, for direct or indirect        detection with a measuring instrument, e.g. an optical,        magnetic, electric or chemiluminescent measuring instrument or        method of measurement,    -   labeling of the oligonucleotides, of the fragments, of        covalently or noncovalently bound hybrid strands constructed        therefrom and of the target sequences, for direct or indirect        detection by other molecular structures such as enzymes,        receptors, proteins,    -   isolation or extraction of the oligonucleotides, of the        fragments, of covalently or noncovalently bound hybrid strands        constructed therefrom and of the target sequences,    -   binding of the oligonucleotides, of the fragments, of covalently        or noncovalently bound hybrid strands constructed therefrom and        of the target sequences by the haptens or other building blocks        that can be used in a molecular recognition reaction, of a        functional group or of a modification,    -   detection of the oligonucleotides, of the fragments, of        covalently or noncovalently bound hybrid strands constructed        therefrom and of the target sequences,    -   decomposition, selective separation, opening, degradation or        enzymatic digestion of the oligonucleotides, of the fragments,        of covalently or noncovalently bound hybrid strands constructed        therefrom and of the target sequences,    -   attachment of the oligonucleotides, of the fragments, of        covalently or noncovalently bound hybrid strands constructed        therefrom and of the target sequences to a target structure or        to target structures, e.g. in or on a reaction support, on a        glass slide, in a reaction vessel, to a target organelle, to a        target cell, to a target organ, to an organism, to a surface, to        a biological surface, to a molecular complex such as a        chromosome, virus particle or protein complex.

An example of application of the invention and the course of the methodusing the GENIOM® platform are presented below:

-   1. Design of a microarray from 6000 different 30-40mer, in    particular 30mer or 40mer nucleic acid fragments in the GENIOM®    equipment. In designing the microarray, the number of synthesis    spots per sequence is chosen between 1 and 100 (in single steps),    with nucleic acid fragments (oligos) with tested or predicted weaker    binding relative to other sequences that participate in assembly    later, being represented with a number of synthesis spots that is    preferably greater by 50% or more, than at least one other    oligo-sequence, in order to increase the percentage in the mixture    and thus promote the corresponding hybrids versus the other,    stronger-binding sequences.-   2. Adding-on of a generic primer-sequence of 10-15, in particular 10    or 15 bases to all 6000 oligos, in particular at both ends in each    case, so that all sequences comprise e.g. 60 bases. The primer    sequence can be selected so that a PCR reaction is possible by    hybridization of in each case two oligos with complementary    sequence.-   3. Production of the microarray based on the design from 1. and 2.    by synthesis in the GENIOM® equipment.-   4. Cleaving-off and elution of the oligos from the reaction support.-   5. PCR of the library with addition of a primer pair suitable for    the primer sequences added in 2. In the PCR there is formation of    e.g. 60mers, each of which can carry an insert of e.g. 40 bases with    optional sequence and uniform sequences at both ends.-   6. Cleaving-off of the primer sequence.-   7. Incubation of the resultant 30-40mer library or of the 30mer or    40mer library with new PCR reagents with addition of a primer pair,    which e.g. only binds to sequences in the hybrid that are at least 2    fragments apart and therefore leads to a “nested PCR” of a longer    fragment (direct assembly and PCR amplification of a synthetic gene    is known by a person skilled in the art from publications).-   8. Further processing or storage of the resultant amplification    product (amplicon). In one embodiment the amplicon is cloned in    bacteria using a plasmid and after growth of clones a number of 10    clones or 10 inserts in the clones is sequenced. The sequence-tested    synthetic gene is ready.

It is known in the prior art that the use of different quantitativeproportions of the individual oligonucleotides for assembly of syntheticgenes increases the rate of correct sequences (Gao, X et al., NucleicAcids Research, 2003, Vol 31; No. 22; p. 143). The stoichiometry of theoligonucleotides has an influence on the thermodynamic parameters on thebasis of the law of mass action.

In the preferred embodiment and in a number of the methods described atthe beginning for the production of microarrays in situ, the design,i.e. the actual loading and allocation of area and location on thereaction support for an individual oligonucleotide species, can beselected flexibly and hence also the quantity of the individualoligonucleotides. Basically this is possible with all the in situmethods of synthesis known by a person skilled in the art and enumeratedat the beginning. Methods that can be programmed flexibly and do notrequire any change of physical parts in the production setup areparticularly preferred for the embodiments of the invention. Forexample, the ink-jet spotting methods, the projection methods and theCombimatrix electrochemical method are particularly advantageous.

The quantity of individual oligonucleotides is correspondingly, afterdetachment and elution of the oligos, also controllable in the pool forvarious oligo-species in solution. When using projection technology forsynthesis on the reaction support, the quantitative distribution can beset via the number of micromirrors or projection elements (illuminationpixels). A person skilled in the art can see from this example that alsoanalogously to other methods, the design determines the quantities. Forexample, in a photolithographic method the quantity is determined by thearea of the synthesis locations, and in an indirect method based on theuse of photo-acids it is determined by the number of physicallyseparated reaction locations that are used for a sequence.

The quantitative proportions can be set and controlled by the user or bysoftware. With suitable software it is possible to program predeterminedvalues for setting the quantitative proportions. In this way the processcan be automated in certain places. The inputs can be derived fromtheoretical models, bioinformatics, sequence comparison, empirical dataor information in databases.

For empirical determination and for finding the optimal number ofsynthesis units per sequence, in a preferred embodiment withmicromirrors in a projection unit, a hybridization reaction can be usedon a microarray. For this, the selected oligos in solution arehybridized on a microarray that contains sequences that are provided forassembly as complementary counterparts on the respective, correspondingoligos. This analysis simulates the assembly reaction. The result can,in one embodiment, be determined with fluorescence markers, which aresecured directly or indirectly to the oligos in solution. The signalsfrom individual analysis spots can then be evaluated relative to oneanother or quantified. As a result of this analysis, the quantitativeproportions can be adjusted by altering the synthesis design.

Adjustment of the quantitative proportions improves the bindingconditions for oligos with increased amount in the mixture, andinherently comparatively lower binding strengths are compensated. Theprobability of correct incorporation in the full set of oligos that takepart in the reaction is equilibrated for all oligos.

A comparatively lower binding strength of an oligo with a suitablesequence on a second oligo can be caused, apart from other parameters,by the base composition, mainly by the AT fraction (if only naturalbases are used), by the tendency to secondary structures or by furtherinteractions with other oligos in the mixture.

In one embodiment, the amplification of the oligonucleotides that aredetached is a component part of the method.

The oligonucleotides or a portion thereof can be synthesized withgeneric 5′- and/or 3′-sequences, added onto the sequence of the nucleicacid that is to be prepared, so that an amplification of theoligonucleotides or of a portion thereof can then take place. Theamplification or primer sequences are complementary to correspondingamplification primers and can contain one or more cleavage sites,preferably Type II cleavage sites. These cleavage sites enablesplitting-off of the primer sequences after amplification, e.g. by PCR.Several pairs of amplification primer sequences can be used on onesupport, to permit a multiplex amplification, e.g. PCR, in anoligonucleotide library. This means that subpopulations of theoligonucleotide fragments can contain specific, but differentamplification sequences or pairs of amplification sequences. Theamplicons can be purified or can be used directly for the amplificationreaction, e.g. for PCR.

In this embodiment, the method can be used for selective amplificationof a subpopulation of the sequences derived from the support. It alsobecomes possible to complete nucleic acid fragments that were not infull-length form after the synthesis. The amplifications can take placedirectly in the microchannels of the support or/and separately from thesupport in a suitable reaction vessel. In this way the quantity ofnucleic acid fragments available for gene synthesis can be increasedsignificantly. Furthermore, the shortened fragments that formed duringthe synthesis, and that may hamper the assembly reaction, are dilutedand are present at negligible concentrations compared with the amplifiedfull-length fragments. Therefore increasing the proportion offull-length fragments in a mixture intended for gene synthesis by prioramplification and hence dilution of shortened synthesis products canalso be a component part of this embodiment.

In yet another—optionally independent—embodiment, nucleic acid fragmentswith the desired correct sequence can be isolated from a mixture ofnucleic acids. For this it is possible for example to use knowntechniques, such as emulsion-based PCR or individual molecular arrays,in which clonal molecular populations or individual molecules can beisolated and sequenced from a mixture of DNA fragments. By using suchtechniques during the gene synthesis method, the assembled gene can be“monoclonalized” and each of the individual fragments can be sequencedseparately. After sequence verification, the fragment with the desiredsequence can be identified, isolated and processed further, e.g. bycloning, DNA based assays, in vitro protein expression etc. The mixtureof DNA fragments can be a PCR product, a ligation product or anoligonucleotide library.

Methods in which an emulsion-based PCR simultaneously still containspreferably in each case a bead or particle in the micelles or aqueouscompartments, are known by a person skilled in the art. In variants thatare particularly optimized and are preferred for the invention, theseare smaller than 1 mm in diameter. For example, the method of thecompany 454, which permits the sequencing of segments in the range 200to 300 bases (as at 2006), is known. In this, DNA is fragmented intosmaller segments and these are then supplemented with uniform linker oradapter sequences by ligation. This mixture is incubated with the beadsdescribed in an emulsion PCR. The primers for the PCR reaction arepresent as solid phase on the beads. The reaction result is amultiplicity of beads, each of which carries clonally only one fragmentfrom the previously fragmented DNA material covalently on the surface.In the next step the beads are immobilized in a reaction support, whichcontains cavities suitable for the beads and their size, and thendetects the sequences on each of the beads in parallel by a so-calledsequencing by synthesis reaction that is known by a person skilled inthe art.

In the method according to the invention or in an optionally independentembodiment, first one or more assembly reactions can be combined fromthe oligos from the parallel synthesis. In one embodiment usingemulsion-bead-PCR, the target sequences are to be selected according tothe reading widths of the sequencing reaction and are thereforepreferably 10 to 1000 nucleotides long, especially preferably 40 to 500.The advantage of this embodiment is that a mixture of assembledsequences, each built up from 2 or more oligos and possibly containingdefects, is, in the method presented above, amplified on the beads toclonal populations and these are then sequenced. Therefore, in apreferred embodiment, cloning and quality control of the targetsequences can be combined in one step. Localization is effected byimmobilization in the support during sequencing. Those DNA targetsequences with sequences that meet a predefined criterion can be removedin a next step. In a further preferred embodiment, labeling is carriedout by spatially-resolved addition of a marker, e.g. a specifically ornonspecifically binding optical marker, such as an intercalator(Sybergreen).

An optionally independent embodiment comprises a method of in particularparallel sequencing of at least one nucleic acid in a mixture comprisingassembled nucleic acids, which possibly contain defective nucleotides,comprising the steps:

-   (a) amplification of a mixture comprising assembled nucleic acids,    which are in each case built up from 2 or more nucleic acid    fragments, to clonal populations,-   (b) sequencing of at least one clonal population from step (a) and-   (c) optionally isolation of at least one nucleic acid, which    contains defects, or/and at least one nucleic acid, which is    correct.

In a preferred embodiment isolation takes place by isolation of one ormore beads. In an alternative embodiment isolation takes place byselective amplification by spatially-resolved addition of PCR reagents.In an alternative embodiment labeling takes place by spatially-resolvedaddition of a marker, e.g. a specifically or nonspecifically bindingoptical marker, such as an intercalator (Sybergreen), and subsequentelution by the laser capture method, which is known by a person skilledin the art from the isolation of individual cells.

Clones (beads) that are undesirable or are recognized as defective canbe eliminated physically. In one embodiment this can take place byselective treatment with a strong light source such as a laser.Alternatively a further immobilization or derivatization can be carriedout, e.g. in a light-dependent reaction, e.g. crosslinking, covalentmodification or the adding-on of a molecule that facilitates extractionor elimination. Thus, beads with an undesirable sequence can beselectively excluded during further exploitation of the sequencedproduct or the plurality of products.

Having been eluted, isolated or otherwise made available for furthersteps, the desired target sequences can be used for building up evenlonger target sequences. They can also be used as a mixture forsubsequent process steps.

In one embodiment, all constituents of a genome that are regarded asnecessary are produced in this method. In a preferred embodiment theseDNA segments are, in a subsequent step, inserted in a target organism,which constructs an assembled genome from them in vivo. An especiallypreferred target organism is Deinococcus radiodurans (also known asMicrococcus radiodurans), which can assemble its own genome in vivoafter fragmentation, e.g. ionizing radiation, into fragments smallerthan 10 000 bases.

The beads can be isolated or stored in the sequencing reaction supportand used again at a later time.

The clonal sequences can be obtained by detachment or copying withoutdisrupting the covalent linkage to the bead. With copying withoutdisrupting the covalent linkage to the bead, a bead is available laterfor the clonal sequences to be obtained again.

Parallel sequencing methods like those described above are suitable forthe verification of mixtures of oligonucleotides, as described as partof the invention and as starting material for gene synthesis. In afurther—optionally independent—embodiment, the composition of a libraryof oligos from a parallel synthesis method such as the method accordingto the invention is verified in a parallel sequencing method with atleast 100, preferably with 1000 to 10 000, especially preferably with 10000 to 100 000 and in particular with 100 000 to 100 million parallelsequencing reactions.

A person skilled in the art knows other sequencing methods that can beused in the present invention, for example the preparation of so-calledpolonies as clonal DNA on a reaction support and subsequent sequencingby “sequencing by synthesis reaction” or by “sequencing by ligation”.Products that use these methods are obtainable from, among others, thecompany Applied Biosystems (ABI, USA) under the name Solid and from thecompany Solexa/Illumina (USA). A special embodiment with especiallysensitive detection is the “true single molecule sequencing” (tSMS) fromthe company Helicos (USA), which also takes place in parallel andtherefore can also be used for the invention.

In combination with the selection of the quantity of synthesis capacity,e.g. illumination pixels, mentioned above, a control or regulating logicsystem can be used as part of the invention. Because the sequencingmethods included here are highly parallel, it is possible to recordlarge amounts of data and therefore provide rational adjustment ofsynthesis parameters, so as to base the proportion of usable targetsequences on defined criteria, e.g. proportion of correct targetsequences.

An—optionally independent—further embodiment of the invention relates tothe production of libraries that contain a multiplicity of variants of agene, through synthesis of multiple variants of one or more of thenucleic acid fragments on the support before gene assembly.

Yet another—optionally independent—embodiment of the invention relatesto the enzymatic cleaving-off of the nucleic acid fragments from thesupport.

Yet another—optionally independent—embodiment of the invention relatesto the purification of a library of nucleic acid fragments throughrehybridization on a support that contains the complementary sequences.

Yet another—optionally independent—embodiment of the invention relatesto the adding-on of primer-specific DNA sequences to the nucleic acidfragments that form the 5′-end and the 3′-end of the nucleic acid doublestrand that is to be synthesized. In this way several successivereactions can be carried out with the same primer pair. Moreover,different genes from different supports can be amplified simultaneouslywith the same primers, whereby the method is automated.

Yet another—optionally independent—embodiment of the invention relatesto the production of synthetic target nucleic acids for standardmicroarray analysis methods, e.g. of probes that are immobilized onstandard microarrays.

The aforesaid embodiments can of course be combined with one another.

In general, all reaction supports and solid phases, for which synthesisof a matrix of nucleic acid polymers is possible, can be used for themethod according to the invention.

These include, as typical representatives, the following reactionsupport formats and solid phases that are known by a person skilled inthe art:

-   -   flat reaction support, also called “chip”,    -   porous supports,    -   reaction supports with electrodes,    -   reaction support with temporarily or permanently immobilized        solid phase of particles or beads,    -   microfluidic reaction support,    -   surface modification: gels, linkers, spacers, polymers,        amorphous layers, 3D matrices.

Some of these reaction supports can be used in combination, e.g. amicrofluidic reaction support with porous surfaces.

The DNA probes are preferably constructed by light-controlled in situsynthesis on a microfluidic support, e.g. in a GENIOM® one instrument(febit biotech GmbH) using suitable protecting-group chemistry in athree-dimensional microstructure. In a cyclic synthesis process,illuminations and condensations of the nucleotides alternate until thedesired DNA sequence has been built up completely in the microchannelsat each position of the array. In this way e.g. up to 48 000oligonucleotides with a length of e.g. up to 60 individual buildingblocks can be prepared. The oligonucleotides can bind covalently to aspacer molecule, a chemical spacer on the glass surface of the reactionsupport. Synthesis takes place under software control and permits highflexibility in the construction of the array, which the user cantherefore configure individually according to his needs. For example,the length of the oligonucleotides, the number of nucleic acid probesproduced or internal controls can be optimized for the particularexperiment.

In one embodiment, high-quality nucleic acids with a freely programmablesequence are prepared in the form of oligonucleotides with a length of10-200 bases, inexpensively and efficiently in a plurality of 10 or moredifferent sequences, in order to produce synthetic codingdouble-stranded DNA (synthetic genes).

The construction of double-stranded DNA from oligonucleotides has beenknown since the 1960s (works of Khorana and others; see “Shabarova:Advanced Organic Chemistry of Nucleic Acids”, VCH Weinheim). In themajority of cases it is carried out by one of two methods (seeHolowachuk et al., PCR Methods and Applications, Cold Spring HarborLaboratory Press).

In one case synthesis of the complete double strand is carried out bysynthesis of single-stranded nucleic acids (of suitable sequence),assembly by hybridization of complementary regions of these singlestrands and ligation of the molecular backbone by enzymes, generallyligase.

Conversely, there is also the possibility of synthesis of regions thatoverlap at the edges as single-stranded nucleic acids, assembly byhybridization, filling-up of the single-stranded regions by enzymes(polymerases) and then ligation of the backbone by enzymes, generallyligase.

A preferred course of gene synthesis according to the invention is asfollows: generally, within the scope of a modular system, manyindividual nucleic acid strands are synthesized using the methodaccording to the invention for highly parallel matrix-based DNAsynthesis. The reaction products are sets of nucleic acids, which serveas building blocks in a subsequent process. As a result, a sequencematrix is produced that can contain more than 100 000 differentsequences. The nucleic acids are in single-stranded form and can beeluted from the support or can be reacted directly in the reactionsupport. By repeated copying in one or more operations, multiple copiesof the matrix can be produced without destroying it, and multiplicationof the particular sequences encoded in the matrix is achieved at thesame time. As described in more detail elsewhere, by copying from distalto proximal it is also possible to cut down the proportion of shortenednucleic acid polymers on the solid phase, if the copying initiation siteis located distally. An example is a distally attached promotersequence.

The support with the matrix of molecules bound to the solid phase can bestored for later reuse. The plurality of sequences produced in onereaction support by an in situ synthesis can thus be made available forfurther process steps. At the same time, through the design of thecopying reaction, high quality of the copied sequences can be achieved.

Then suitable combinations of the detached DNA strands are formed.Assembly of the single-stranded building blocks to double-strandedbuilding blocks takes place within a reaction space, which in a simplesetup can be an ordinary reaction vessel, e.g. a plastic tube. Inanother preferred embodiment the reaction space is part of the reactionsupport, which in one variant can be a microfluidic reaction support, inwhich the necessary reactions take place. A further advantage of anintegrated microfluidic reaction support is the possibility ofintegration of further process steps, such as quality control by opticalanalysis. In one embodiment the matrix has already been synthesized in amicrofluidic support, which can then be used simultaneously as reactionspace for the subsequent assembly.

The sequence of the individual building blocks is selected so that onbringing the individual building blocks in contact with one another,complementary regions at the two ends brought together are available topermit specific assembly of DNA strands through hybridization of theseregions. This results in longer DNA hybrids. The phosphodiester backboneof the DNA molecule is closed by ligases. If the sequences are selectedin such a way that there are single-stranded gaps in these hybrids,these gaps are filled enzymatically by polymerases in a known procedure(e.g. Klenow fragment or Sequenase). This results in longerdouble-stranded DNA molecules. If further use requires these elongatedDNA strands to be available in the form of single strands, this can beachieved by the methods that are known to a person skilled in the artfor denaturing DNA double strands, such as temperature or alkali.

By bringing together clusters of DNA strands synthesized in this waywithin reaction spaces, it is once again possible to produce longerpartial sequences of the final DNA molecule. This can be carried out instages, and the partial sequences are then assembled to longer andlonger DNA molecules. In this way it is possible to produce very longDNA sequences as a completely synthetic molecule with a length of morethan 100 000 base pairs. This already corresponds to the order ofmagnitude of a bacterial artificial chromosome (BAC). Construction of asequence of 100 000 base pairs from overlapping building blocks of 20nucleotides length requires 10 000 individual building blocks.

This can be accomplished with most of the highly parallel methods ofsynthesis described at the beginning. Those technologies that producethe array of nucleic acid polymers in a largely freely programmablemanner, and do not rely on the setting-up of technical components, suchas photolithographic masks, are especially preferred for the methodaccording to the invention. Accordingly, especially preferredembodiments employ projector-based light-based synthesis, indirectprojector-based light-controlled synthesis by means of photo-acids andreaction chambers in a microfluidic reaction support, electronicallyinduced synthesis by spatially-resolved deprotection on individualelectrodes on the support and fluidic synthesis by spatially-resolveddeposition of the activated synthesis monomers.

For the rational processing of genetic molecules and the systematicacquisition of all possible variants, the building blocks must beproduced flexibly and economically in their individual sequence. Themethod accomplishes this through the use of a programmable light sourcematrix for the light-dependent spatially-resolved in situ synthesis ofthe DNA strands that are used as building blocks. This flexiblesynthesis permits the free programming of the individual sequences ofthe building blocks and hence also the production of any variants of thepartial sequences or of the final sequence, without any associatedsubstantial modifications of system components (hardware). Only thisprogrammed synthesis of the building blocks and hence of the finalsynthesis products can provide systematic processing of the plurality ofgenetic elements. At the same time, the use of computer-controlledprogrammable synthesis makes it possible to automate the entire processincluding communication with corresponding databases.

When the target sequence is specified, the sequence of the individualbuilding blocks can be selected rationally taking into accountbiochemical and functional parameters. Following input of the targetsequence (e.g. from a database) an algorithm searches for the suitableoverlapping regions. Depending on the definition of the task, varyingnumbers of partial sequences can be set up, namely within one reactionsupport that is to be illuminated or distributed on several reactionsupports. The attachment conditions for formation of the hybrids, suchas temperature, salt concentration etc., are matched by a correspondingalgorithm to the available overlapping regions. This ensures maximalspecificity of assembly. The data for the target sequence can, in afully-automatic version, also be taken directly from public or privatedatabases and converted to corresponding target sequences. The resultantproducts can, once again optionally, be fed into appropriately automatedprocesses, e.g. into cloning in suitable target cells.

Construction in stages by synthesis of the individual DNA strands inreaction regions within circumscribed reaction spaces also makes itpossible to construct difficult sequences, e.g. those with internalrepeats of sequence segments, such as are found e.g. in retroviruses andcorresponding retroviral vectors. By detaching the building blockswithin the fluidic reaction spaces, any sequence can be synthesized,without problems arising from allocation of the overlapping regions onthe individual building blocks.

The high quality requirements that are necessary when constructing verylong DNA molecules are fulfilled inter alia through the use of real-timequality control. The spatially-resolved synthesis of the building blocksis monitored, as is the detachment and assembly as far as constructionof the final sequence. Then all processes take place in a transparentreaction support. It is, moreover, possible to monitor reactions andfluidic processes in transmitted light, e.g. using CCD detection.

The miniaturized reaction support is designed so that a detachmentprocess is possible in the individual reaction spaces, and therefore theDNA strands synthesized on the reaction regions within these reactionspaces are detached in clusters. With suitable design of the reactionsupport, assembly of the building blocks can take place in stages inreaction spaces, as well as the removal of building blocks, partialsequences or of the final product, or also sorting or separation of themolecules.

Once it is completed as an integrated genetic element, the targetsequence can be inserted in cells by transfer and can thus be cloned andcan be investigated in the course of functional studies. Anotherpossibility is first to carry out further purification or analysis ofthe synthesis product, whereas said analysis can for example besequencing. The sequencing process can also begin by direct couplingwith suitable equipment, e.g. with a device operating according topatent application DE 199 24 327 for integrated synthesis and analysisof polymers. It is also conceivable for the target sequences produced tobe isolated and analyzed after cloning.

The method according to the invention provides, with the integratedgenetic elements that it produces, a tool that encompasses thebiological variety in a systematic process for the further developmentof molecular biology. The production of DNA molecules with desiredgenetic information is therefore no longer the bottleneck in molecularbiological work, because from small plasmids via complex vectors and asfar as mini-chromosomes, all molecules can be produced synthetically andmade available for further work.

The method of preparation permits the parallel production of numerousnucleic acid molecules and therefore a systematic approach for questionsconcerning regulatory elements, DNA binding sites for regulators, signalcascades, receptors, action and interactions of growth factors etc.

Through integration of genetic elements in a completely synthetic totalnucleic acid, the known genetic tools, such as plasmids and vectors, cancontinue to be used, building on relevant experience. On the other handthis experience will change rapidly through the endeavours to optimizethe existing vectors etc. The mechanisms that for example make a plasmidsuitable for propagation in a particular cell type can be investigatedrationally for the first time on the basis of the method according tothe invention.

Through this rational investigation of large numbers of variants, theentire combination space of genetic elements can be opened up. Alongwith highly parallel analysis (including on DNA arrays or DNA chips)that is currently undergoing rapid development, the programmed synthesisof integrated genetic elements is created as a second important element.Only both elements together can form the foundation of rationalmolecular biology.

With the programmed synthesis of corresponding DNA molecules, it is notonly possible to have any desired composition of coding sequences andfunctional elements, but also the regions in-between can be adapted.This should quickly lead to minimal vectors and minimal genomes, onceagain producing advantages through the smaller size. Transmissionvehicles, such as viral vectors, can thus be made more efficiently, e.g.using retroviral or adenoviral vectors.

Beyond the combination of known genetic sequences, the development ofnew genetic elements is possible, which can build on the function ofexisting elements. It is precisely for such development work that theflexibility of the system is of enormous value.

The synthetic DNA molecules are fully compatible, at every stage ofdevelopment of the method described here, with existing recombinanttechnology. Integrated genetic elements can also be provided for“traditional” molecular biological applications, e.g. by means ofappropriate vectors. The incorporation of corresponding cleavage siteseven for enzymes that have so far found little application is not alimiting factor for integrated genetic elements.

This method makes it possible to integrate all desired functionalelements as “genetic modules”, such as genes, parts of genes, regulatoryelements, viral packaging signals etc., into the synthesized nucleicacid molecule as carrier of genetic information. This integration offersthe following advantages, among others:

High-grade functionally integrated DNA molecules can be developed,omitting unnecessary DNA regions (minimal genes, minimal genomes).

Free combination of the genetic elements and the changes to thesequence, e.g. for adaptation to the expressing organism/cell type(codon usage), are also made possible, as are changes to the sequencefor optimizing functional genetic parameters, for example generegulation.

Changes to the sequence for optimizing functional parameters of thetranscript also become possible, e.g. splicing, regulation at the mRNAlevel, regulation at the translation level, and furthermore theoptimization of functional parameters of the gene product, for examplethe amino acid sequence (e.g. antibodies, growth factors, receptors,channels, pores, transporters etc.).

Furthermore, it is possible to devise constructs that interfere withgene expression by the RNAi mechanism. When such constructs encode morethan one RNAi species, several genes can be inhibited simultaneously ina multiplex system.

Overall, the system achieved with the method is extremely flexible andpermits, in a manner not previously available, the programmedconstruction of genetic material at greatly reduced expenditure of time,materials and work.

Directed manipulation of larger DNA molecules, such as chromosomes ofseveral hundred kbp, was practically impossible with the existingmethods. In fact, more complex (i.e. larger) viral genomes of more than30 kbp (e.g. adenoviruses) are difficult to handle and manipulate withthe classical methods of genetic engineering.

There is considerable shortening as far as the last stage of cloning ofa gene: the gene or genes are synthesized as a DNA molecule and then(after suitable preparation, such as purification etc.) are inserteddirectly into target cells and the result is investigated. Themultistage cloning process, generally taking place via microorganismssuch as E. coli (e.g. DNA isolation, purification, analysis,recombination, cloning in bacteria, isolation, analysis etc.), istherefore reduced to the last transfer of the DNA molecule into thefinal effector cells. With synthetically produced genes or genefragments, clonal multiplication in an intermediate host (generally E.coli) is no longer necessary. We thus avoid the risk that the geneproduct intended for the target cell has a toxic action on theintermediate host. This is a clear contrast from the toxicity of somegene products, which when using classical plasmid vectors often leads toconsiderable problems in cloning the corresponding nucleic acidfragments.

Another appreciable improvement is the shorter time and the reduction inprocess steps, until, after sequencing of the genetic material, theresultant potential genes are verified as such and are cloned. Normallyafter discovering interesting patterns, which may be considered as ORF,corresponding clones are sought with probes (e.g. by PCR) in cDNAlibraries, though they need not contain the entire sequence of themessenger RNA (mRNA) used originally in their production (the problem of“full length clones”). In other methods, an antibody is used forsearching in an expression gene library (screening). Both methods can beshortened considerably with the method according to the invention: if wehave a gene sequence that has been determined “in silico” (i.e. afterrecognition of a corresponding pattern in a DNA sequence by thecomputer), or after decoding a protein sequence, a corresponding vectorwith the sequence or variants thereof can be produced directly viaprogrammed synthesis of an integrated genetic element and inserted insuitable target cells.

The synthesis of DNA molecules in this way up to several 100 kbp permitsthe direct complete synthesis of viral genomes, e.g. adenoviruses. Theseare an important tool in basic research (including gene therapy), butbecause of the size of their genome (approx. 40 kbp) they are difficultto manipulate with classical methods of genetic engineering. Inparticular this greatly limits the rapid and economical production ofvariants for optimization. This limitation is removed by the methodaccording to the invention.

With the method, synthesis, detachment of the synthesis products andassembly to a DNA molecule are integrated in one system. With theproduction methods of microsystem engineering, all necessary functionsand process steps up to purification of the final product can beintegrated in a miniaturized reaction support. These can comprisesynthesis regions, detachment regions (clusters), reaction spaces, feedchannels, valves, pumps, concentrators, separation regions etc.

Plasmids and expression vectors can be produced directly for sequencedproteins or corresponding partial sequences and the products analyzedbiochemically and functionally, e.g. using suitable regulatory elements.The search for clones in a gene library is therefore omitted.Correspondingly, open reading frames (ORF) from sequencing work (e.g.the human genome project) can be programmed directly in correspondingvectors and combined with desired genetic elements. Identification ofclones, e.g. by costly screening of cDNA libraries, is unnecessary. Theflow of information from sequence analysis to function analysis istherefore much shorter, because on the same day that an ORF is in thecomputer from analysis of primary data, a corresponding vector includingthe presumed gene can be synthesized and made available.

Relative to conventional solid-phase synthesis for obtaining syntheticDNA, the method according to the invention is characterized by lowerexpenditure of materials. For the production of thousands of differentbuilding blocks for the production of a complex integrated geneticelement with length of several 100 000 kbp, in correspondinglyparallelized format and with corresponding miniaturization (see Examplesof application), a microfluidic system uses far less feed material thana conventional automatic machine for solid-phase synthesis for anindividual DNA oligomer (when using a single column). Here, microlitersare contrasted with the consumption of milliliters, i.e. a factor of1000.

Taking into account the latest findings in immunology, the methoddescribed permits extremely efficient and rapid vaccine design (DNAvaccines).

1. A method for isolating an individual nucleic acid molecule having adesired correct sequence from a mixture of nucleic acids, comprising:(a) monoclonizing a mixture of nucleic acids on a solid reactionsupport; (b) parallel sequencing the monoclonized nucleic acids of step(a), (c) localizing a nucleic acid molecule with the desired correctsequence in the nucleic acids of step (b), (d) isolating the nucleicacid molecule from step (c), and (e) optionally further processing ofthe isolated nucleic acid molecule obtained in step (d).
 2. A method forisolating an individual nucleic acid having a desired sequence from amixture of nucleic acids, comprising: (a) monoclonizing a mixture ofnucleic acids on a solid reaction support; (b) parallel sequencing themonoclonized nucleic acids of step (a), (c) localizing a nucleic acidwith the desired sequence in the nucleic acids of step (b), (d)isolating the nucleic acid from step (c), and (e) optionally furtherprocessing of isolated nucleic acid obtained in step (d) wherein theisolation takes place by at least one of steps (i) to (iv): (i)isolating one or more beads on which said nucleic acid molecule isimmobilized, (ii) cleaving off the nucleic acid molecule from a supporton which said nucleic acid molecule is immobilized, (iii) selectivelyamplifying the nucleic acid molecule by spatially-resolved addition ofPCR reagents, (iv) eluting the nucleic acid molecule by laser capturemethod.
 3. A method for isolating an individual nucleic acid moleculehaving a desired correct sequence from a mixture of nucleic acids,comprising: (a) monoclonizing a plurality of the different nucleic acidsby emulsion-based PCR on beads (b) immobilizing the beads in a reactionsupport (c) parallel sequencing the monoclonized nucleic acids of step(a) (d) localizing an individual nucleic acid molecule with the desiredcorrect sequence in the nucleic acids of step (c) (e) isolating thebeads comprising the nucleic acid molecules localized in step (d) (f)obtaining the clonal nucleic acid sequences by detachment of the clonalnucleic acid sequence from the bead or by copying (f) optionally furtherprocessing of the isolated nucleic acid molecule obtained in step (d).4. A method for quality control of assembled nucleic acid molecules,said method comprising: (a) providing a mixture of assembled nucleicacid molecules suspected of containing sequence defects, (b) amplifyingthe mixture of (a) to receive a clonal population thereof, (c)immobilizing the clonal population of (b) on a support, (d) parallelsequencing the clonal population immobilized according to (c), (e)localizing at least one clonal nucleic acid molecule that meets apredefined criterion (f) obtaining the at least one clonal nucleic acidmolecule of (e).
 5. A method for isolation of a sequence-verifiednucleic acid clone, comprising: (a) providing a mixture of nucleic acidmolecules suspected of containing sequence defects, (b) amplifying themixture of (a) to receive a clonal population thereof, (c) immobilizingthe clonal population of (b) on a support, (d) parallel sequencing theclonal population immobilized according to (c) (e) localizing at leastone clonal nucleic acid molecule that meets a predefined criterion, and(f) obtaining the at least one clonal nucleic acid molecule of (e). 6.The method of claim 4, wherein the assembled sequences provided in step(a) are built each from one or more oligonucleotides.
 7. The method ofclaim 4, wherein the assembled sequences represent genes, gene clusters,chromosomes, genomes or fragments thereof.
 8. The method of claim 4,wherein amplification in step (b) comprises an emulsion-based PCR. 9.The method of claim 4, wherein immobilizing the clonal populationaccording to step (c) comprises immobilization on beads.
 10. The methodof claim 4, wherein the predetermined criterion in step (e) is thedesired correct sequence.
 11. The method of claim 4, wherein theobtaining the clonal nucleic acid molecule of (f) comprises at least oneof: (i) detachment of the clonal nucleic acid molecule from the support,(ii) copying of the clonal nucleic acid molecule, (iii) laser capture,or/and (iv) amplification by spatially resolved PCR.
 12. A method ofassembly of nucleic acid molecules comprising: (i) providingsingle-stranded nucleic acid molecules to be assembled into a targetnucleic acid molecule, (ii) assembling by hybridization complementaryregions of the single-strands nucleic acids of (i), (iii) controllingthe quality of the mixture of assembled nucleic acid molecules obtainedin step (ii) by the method of claim
 4. 13. The method of claim 12,wherein step (ii) results in a gene, gene cluster, chromosome, genome ora fragment thereof.
 14. The method of claim 1 wherein the furtherprocessing comprises assembling nucleic acid molecules of step (d) to adesired nucleic acid.
 15. The method of claim 1 wherein the solidreaction support is selected from the group consisting of: one or morebeads, one or more particles, one or more chips, and a microfluidicreaction support.
 16. The method of claim 15 wherein the solid reactionsupport is one or more beads or a chip.
 17. The method of claim 5wherein the solid reaction support is selected from the group consistingof: one or more beads, one or more particles, one or more chips, and amicrofluidic reaction support.
 18. The method of claim 17 wherein thesolid reaction support is one or more beads or a chip.